Role of Oxidative Stress in the Pathogenesis of Atherothrombotic Diseases

Oxidative stress is generated by the imbalance between reactive oxygen species (ROS) formation and antioxidant scavenger system’s activity. Increased ROS, such as superoxide anion, hydrogen peroxide, hydroxyl radical and peroxynitrite, likely contribute to the development and complications of atherosclerotic cardiovascular diseases (ASCVD). In genetically modified mouse models of atherosclerosis, the overexpression of ROS-generating enzymes and uncontrolled ROS formation appear to be associated with accelerated atherosclerosis. Conversely, the overexpression of ROS scavenger systems reduces or stabilizes atherosclerotic lesions, depending on the genetic background of the mouse model. In humans, higher levels of circulating biomarkers derived from the oxidation of lipids (8-epi-prostaglandin F2α, and malondialdehyde), as well as proteins (oxidized low-density lipoprotein, nitrotyrosine, protein carbonyls, advanced glycation end-products), are increased in conditions of high cardiovascular risk or overt ASCVD, and some oxidation biomarkers have been reported as independent predictors of ASCVD in large observational cohorts. In animal models, antioxidant supplementation with melatonin, resveratrol, Vitamin E, stevioside, acacetin and n-polyunsaturated fatty acids reduced ROS and attenuated atherosclerotic lesions. However, in humans, evidence from large, placebo-controlled, randomized trials or prospective studies failed to show any athero-protective effect of antioxidant supplementation with different compounds in different CV settings. However, the chronic consumption of diets known to be rich in antioxidant compounds (e.g., Mediterranean and high-fish diet), has shown to reduce ASCVD over decades. Future studies are needed to fill the gap between the data and targets derived from studies in animals and their pathogenetic and therapeutic significance in human ASCVD.


Introduction
Oxidative stress is generally defined as an imbalance between formation of reactive oxygen species (ROS) [1] and their clearance by antioxidant systems [2]. ROS include molecules and free radicals (i.e., chemical species with one unpaired electron) derived from molecular oxygen (O 2 ) formed in the cell cytoplasm, endoplasmic reticulum (ER), mitochondria, peroxisomes [3,4] and extracellular space ( Figure 1).
While O2 by itself is not very reactive, if one of its unpaired electrons is excited, the resulting species become powerful oxidants [5]. Superoxide anion (O2 •− ), is the precursor of most ROS, such as hydrogen peroxide (H2O2), which may then generate the hydroxyl radical ( • OH) and the peroxynitrite (ONOO − ) by reacting with nitric oxide (NO) [4] (Figure 1). O2 •− can be produced during enzymatic reactions, e.g., by cytochrome P450, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), or xanthine oxidase (XO) in the cell cytoplasm [2]. O2 •− can also be non-enzymatically released along the mitochondrial electron transport chain (ETC) reactions, especially by complexes I and III [3,4] (Figure 1). Depending on their origin, type and environment, ROS-triggered signals may contribute to both cell homeostasis [3,6] or dysfunction by the non-specific damage of proteins, lipids, nucleic acids, and polysaccharides [4].

Figure 2. ROS scavenger systems in different cell compartments. O2
•− is converted to H2O2 by superoxide dismutases (SODs), SOD1 in the cytoplasm, SOD2 in the mitochondria, and peroxisome, and SOD3 in the extracellular space. Catalase (Cat) catalyzes the reduction from H2O2 to O2 and H2O in mitochondria and peroxisome. Glutathione peroxidases (GPX) catalyze the reduction in H2O2; during the reaction, glutathione (GSH) is converted to its oxidized form (GSSG), which has a decreased ability to reduce peroxide. Once oxidized, GSH can be regenerated from GSSG by the enzyme glutathione reductase (GR) using reduced nicotinamide NADPH as the electron donor. During the process, NADPH is oxidized to NADP + . Peroxiredoxins (PRDX) reduce H2O2 to H2O by utilizing electrons from NADPH via thioredoxin (Trx) and thioredoxin reductase (TR). Paraoxonase (PON) isoforms 2 and 3 can prevent mitochondrial O2 •− generation. Abbreviations: GRX: Glutaredoxin; XO: Xanthine Oxidase. SOD2 is mitochondrial, while SOD1 and 3 are cytoplasmic and extracellular, respectively [8]. Catalase (Cat) is a peroxisome scavenger enzyme, converting H2O2 into H2O and O2 [8] (Figure 2).
Several pre-clinical data suggest that ROS contribute to atherosclerosis through endothelial cell (EC) dysfunction, platelet activation and vascular remodeling [9] (Figure 3), while the translation of pre-clinical evidence into human atherosclerotic cardiovascular disease (ASCVD) seems more complex and less clear. The present review will revise preclinical, clinical and intervention evidence of ROS involvement in atherosclerosis development and its thrombotic complications. superoxide dismutases (SODs), SOD1 in the cytoplasm, SOD2 in the mitochondria, and peroxisome, and SOD3 in the extracellular space. Catalase (Cat) catalyzes the reduction from H 2 O 2 to O 2 and H 2 O in mitochondria and peroxisome. Glutathione peroxidases (GPX) catalyze the reduction in H 2 O 2 ; during the reaction, glutathione (GSH) is converted to its oxidized form (GSSG), which has a decreased ability to reduce peroxide. Once oxidized, GSH can be regenerated from GSSG by the enzyme glutathione reductase (GR) using reduced nicotinamide NADPH as the electron donor. During the process, NADPH is oxidized to NADP + .  [8] (Figure 2).

ROS Generation
Animal models supporting the contribution of ROS in atherosclerosis are summarized in Table 1. Table 1. ROS production and atherosclerosis in animal models and in human diseases.
In conclusion, genetically modified animal models show that several enzymatic and non-enzymatic reactions that generate ROS can contribute to different phases of atherosclerosis. Human evidence on the same enzymes is more limited and often inconsistent.

Scavenger Systems
Studies on ROS scavenger systems are summarized in Table 2. Table 2. Scavenger systems and atherosclerosis in animal models and in human diseases.
Two mammalian ubiquitous Trx isoforms are known ( Figure 2): Trx1 is a cytosolic and nuclear protein, whereas Trx2 is mitochondrial [106]. The Trx-related system reduces oxidized cysteine by interacting with the redox-active center of Trx (Cys-Gly-Pro-Cys), which, in turn, can be reduced by Trx reductase and NADPH [107] (Figure 2). EC-targeted Trx2 +/+ mice show increased scavenging activity for H 2 O 2 and O 2 •− [72], ApoE −/− /Trx2 +/+ mice show improved EC function and reduced atherosclerosis [72] and mice with targeted cardiac Trx2 −/− exhibit high oxidative status and vascular lesions [73,74] (Table 2). Trxs are expressed in human VSMCs of normal coronary arteries and are increased in atherosclerotic coronary arteries from autopsies, especially in macrophages [90] (Table 2), suggesting a possible role of Trx in the protection of human coronary arteries.
MDA is a highly reactive dialdehyde generated from ROS-mediated lipid degradation ( Figure 1) [157]. It can induce protein adducts and cross-linking [158], and is measurable in human blood [159]. Consistent with its lipid origin, plasma MDA and 8-epi-PGF 2α have been shown to be highly correlated in some studies (Table 3) [160]. MDA levels are increased in cigarette smoking [137,139], DM [134], CAD [135,138] patients, and they independently predicted MI and revascularization in CAD patients enrolled in the Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial [136] (Table 3).
Ox-LDLs are the end-product of non-enzymatic O 2 •− modifications ( Figure 1) to both LDL proteins and lipids and are measurable in human plasma [161,162]. Ox-LDLs contribute to foam cell development in the vessel wall and bind to macrophages via scavenger receptors [163] and to ECs through the lectin-like oxidized LDL receptor-1, increasing adhesion molecule binding [164] and platelet activation via the scavenger CD36 receptor [165] ( Figure 3). Enhanced circulating ox-LDLs are reported in acute MI [140]. A meta-analysis of 8644 subjects with or without previous ASCVD showed that increased ox-LDLs are associated with ASCVD recurrence [144]; they also independently predicted carotid and femoral atherosclerosis and ASCVD in a prospective population-based survey of from 40to 79-year-old men and women followed over 10 years [142]. They independently predicted CV death, MI, and angina in 238 CAD patients over 52 months [141], and predicted MI and CV death in acute coronary syndrome (ACS) patients [143] (Table 3).
Protein oxidation can be measured by nitrotyrosine derived from tyrosine nitration, ONOO − and NO, in serum, plasma, and urine samples [166,167]. In a case-control study with 100 CAD patients, circulating nitrotyrosine levels were higher in CAD vs. non-CAD patients, and the rates of CAD and atherosclerosis were increased in the higher nitrotyrosine quartiles [146]. Nitrotyrosine is increased in T2DM patients as compared to healthy subjects [145] (Table 3).
Protein carbonyls, the most frequent ROS-induced protein modification, are markers of the irreversible damage of lysine (Lys), arginine (Arg), proline (Pro), and threonine (Thr) residue oxidation [168], in a process named "primary protein carbonylation". The end-product 2,4-dinitrophenylhydrazine [169,170] is stable and measurable in plasma [171]. Elevated circulating protein carbonyls were detected in T2DM [148,152], in hypercholesterolemia [151], and in CAD patients [149] (Table 3). Advanced glycation end products (AGEs) are protein carbonyls generated in the "secondary protein carbonylation" process through glycoxidation, and N ε -(carboxymethyl)lysine is the most abundant AGE [172], which is measurable in organic fluids and tissues [173]. AGEs cause cell damage by binding its receptor (RAGE), which activates nuclear factor-kappa B (NF-κB) [174], and seem to be involved in T2DM-related CV complications [147,150,153,155]. In a meta-analysis of seven prospective observational studies, including 3718 participants, increased circulating AGEs were associated with increased all-cause and CV mortality [156] (Table 3).

Antioxidant Compounds
Several molecules with antioxidant properties have been studied in animal models of atherosclerosis and in humans (Figure 4).
Melatonin appears to increase the activity of antioxidant enzymes such as SOD and GPX, through Sirtuin (SIRT)-3 [175]. Resveratrol is a phytoalexin derived from grapes [176], likely acting via several mechanisms: the downregulation of NOX expression and activity, mitochondrial O 2 •− reduction [177,178], and increased PON1 activity ( Figure 4A). Vitamin E refers to a group of 8 different compounds, 4 tocopherols, and 4 tocotrienols, exerting their antioxidant action by scavenging lipid peroxyl radicals through hydrogen donation from the phenolic group of the chromanol ring ( Figure 4C). Vitamin E inhibits peroxyl radicals before they react with lipids such as cholesterol, cholesterol esters, fatty acids, and phospholipids [179]. Different Vitamin E forms, with the un-substituted 5-position or with the methyl-group in five positions, can also trap reactive NO species [180,181]. Vitamin D inhibits NOX, upregulates several scavenging systems, such as SOD, GPX, and Cat [182] ( Figure 4A), increases NO and the activation of phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) [183] (Figure 4B). Ascorbic acid, i.e., Vitamin C, appears to exert diverse anti-oxidant effects [184] through the inhibition of NOX and XO, SOD activation [185]. Ascorbic acid can preferentially regenerate the Vitamin E radical, while the ascorbic acid radical can be regenerated by GSH [186,187] (Figure 4C). Vitamin B6 is water-soluble; its active form is a cofactor [188], which catalyzes homocysteine trans-sulphuration, contributing to the homocysteine production required for GSH synthesis [189], and is involved in GPX synthesis [190] (Figure 4A). Alpha-lipoic acid (ALA) and its reduced form can regenerate anti-oxidant molecules such as GSH, Vitamin C, Vitamin E, and cofactor Q10 (CoQ10) [191] (Figure 4). Stevioside, a common sweetener [192], contains polyphenol, can increase intracellular reduced GSH, upregulates SOD and Cat and decreases lipid peroxidation [193] (Figure 4A). Acacetin is a natural flavone of plant pigments [194] and can increase SOD2 [195], and Trx activity [196] (Figure 4A). N-3 polyunsaturated fatty acids (n3-PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), seem to have different effects: in mitochondria, DHA reduces the cytochrome complex IV activity and increases SOD [197]. PUFAs upregulate the Nrf-2 transcription that leads to antioxidant gene expression [198] and enhances NO synthesis in ECs [199] (Figure 4A). Some dietary habits appear to be associated with antioxidant properties such as fish consumption, which is likely related to high PUFAs content [200], and some fish proteins also have a scavenger effect by inhibiting lipid peroxidation [201] ( Figure 4C). The Mediterranean diet is rich in green vegetables, fish, and fruit, containing polyphenolic compounds, and PUFAs [202], including nuts and virgin olive oil, which can increase PON-1 activity, reducing lipid peroxidation [203] (Figure 4C).

Studies in Animals
In streptozotocin (STZ)-treated rats that develop DM, the supplementation of melatonin (20 mg/kg once daily (od) per os) for 8 weeks could recover Notch homolog-1 translocation associated/hairy and enhancers of split/protein kinase B (Notch1/Hes/Akt) signal in an I/R injury model and enhanced SOD in aortic VSMC [204] (Figure 4B). In the same animal model, intraperitoneal melatonin (10 mg/kg od) increased SOD and decreased MDA in erythrocytes [205,206] (Figure 4A). 3).

Antioxidant Compounds
Several molecules with antioxidant properties have been studied in animal models of atherosclerosis and in humans (Figure 4).  In DM-induced KsJ-db/db mice, resveratrol added to the chow (0.3% w/w) reduced adhesive molecule expression in aortic ECs [207]. In STZ-DM LDLr −/− mice, resveratrol added to HFD (0.2% w/w) decreased monocyte MCP-1-dependent activation in the aortic root [208]. In ApoE −/− mice, resveratrol (10 mg/kg od) for 6 weeks decreased macrophage differentiation, increased monocytes GSH and decreased atherosclerosis [209]. In C57BL/6 mice on HFD, resveratrol (10 mg/kg in the chow) could restore the integrity of aortic media and recover EC function through the phosphorylation of the Akt/eNOS pathway [210].
Vitamin E (100 mg/od) halved the mortality of HFD-fed mice, and decreased macrophages in atherosclerotic lesions and circulating MDA [211].
Compared to ApoE −/− mice fed with corn oil, ApoE −/− mice fed with fish oil containing n-3 PUFA (32.5 g/100 g total fatty acids) and n-6 PUFA (9.6 g/100 g total fatty acids) reduced atherosclerotic lesions, increased liver GSH and Cat levels [215] and lowered P-selectin and VCAM-1 expression in aorta [216]. Moreover, ApoE −/− mice fed with n3-PUFA-enriched diet had a higher expression of eNOS and reduced O 2 •− in the aorta versus a corn-oil-enriched diet [217]. The supplementation of a western diet with 5% EPA to LDLr −/− mice was associated with lower macrophages' infiltration in the aorta [218]. In HFD-fed ApoE −/− mice, the antioxidant mitoquinone, a ubiquinone analogue, reduced DNA damage and atherosclerotic lesions [219].

Intervention Studies in Humans
Several studies in humans investigated a possible benefit of antioxidants by using biomarkers known as surrogates of either CV protection or CV events, which are summarized in Tables 4 and 5, respectively.
In a small, double-blind, placebo-controlled, randomized clinical trial (RCT) in 60 DM subjects with CAD, supplementation with melatonin (10 mg od) for 12 weeks increased plasma GSH, NO, and decreased MDA and C-reactive protein (CRP) vs. placebo [220] ( Table 4). Table 4. Randomized clinical trials and meta-analyses of antioxidant compounds and dietary intervention on cardiovascular functional surrogates or oxidativestress biomarkers.
Vitamin E (400 UI/od for 24 weeks) supplementation in 187 T2DM subjects did not modify vascular motility or ROS generation [230] (Table 4). A meta-analysis on supplementation with either Vitamin C or E in 296 subjects with T2DM did not show any difference in EC-dependent vasodilation as compared to placebo [222] (Table 4). However, the supplementation of Vitamin E 100 or 600 mg/od for 14 days in 22 hypercholesteremic patients was associated with a dose-dependent, significant decrease in urinary 8-epi-PGF 2α [125]. A systematic review and meta-analysis of 1129 subjects showed a positive effect of Vitamin C on EC-dependent flow-mediated dilation, forearm blood flow, and pulse wave analysis (Table 4) [221]. Notably, the positive effect of Vitamin C was observed in healthy subjects, in whom EC dysfunction was induced by glucose, methionine and endotoxins, and a very high dose of Vitamin C (2600 mg) was used [221]. A meta-analysis of the effect of 33 placebo-controlled RCTs on 1053 DM participants showed that Vitamin D supplementation (between 200 UI/od to 50,000 UI/monthly), was associated with decreased serum CRP and MDA, and increased circulating markers of NO and GSH [225] (Table 4).
While some studies using biomarkers or indirect indexes of CV diseases showed some effect of the antioxidant compounds, RCTs with hard endpoints were largely negative. The Women's Health Study randomized 39,000 healthy women taking Vitamin E (600 UI every other day (eod)) or placebo and failed to show any reduction in MI, stroke or CV death over a mean of 10.1 years [232] (Table 5). Similarly in the Heart Outcomes Prevention Evaluation (HOPE) RCT, Vitamin E (400 UI/ od) did not reduce MI, stroke, and CV death in 9541 subjects with a previous CV event or DM over 4.5 years [234] (Table 5). The Physicians' Health Study II RCT studied a combination of Vitamin E (400 IU/eod) and C (500 mg/od) on MI, stroke, and CV death in 14,641 healthy US male physicians over 8 years, but no benefit was observed versus placebo [238] ( Table 5). The Women's Antioxidant Cardiovascular Study tested Vitamin E (600 IU/od), C (500 mg/od), and beta-carotene (50 mg/eod) on the prevention of MI, stroke, coronary revascularization, or CV death in 8171 women with a history of ASCVD or at least three CV risk factors and failed to show any benefit [237] (Table 5).
A meta-analysis of RCT on the supplementations on Vitamin A, E, C, beta-carotene, and selenium suggested that the some compounds could even increase all-cause mortality, while selenium and ascorbic acid had no effect [245]. The Vitamin D and omega-3 Trial investigated vitamin D cholecalciferol (2000 IU/od) and n-3 FA (1 g/od) on the prevention of MI, stroke, or CV death versus placebo over 5.3 years, showing no benefit [242] (Table 5).
A Study of Cardiovascular Events in Diabetes (ASCEND) RCT randomized n-3 fatty acid (1 g/od) vs. placebo, in >15,000 DM subjects with no evidence of symptomatic CV diseases, and there was no CV benefit associated with omega-3 over 7.4 years [240] ( Table 5). Recently, a meta-analysis including 38 RCTs demonstrated that supplementation with EPA (from 1.8 to 4.0 g/od), or with a combination of EPA and DHA (0.4 to 5.5 g/od), was associated with a reduction in CV mortality, non-fatal MI, and CHD, with a higher reduction observed with EPA monotherapy [243]. However, results regarding the effect of EPA and DHA combination were not confirmed by the same authors when older trials with suboptimal statin therapy were excluded from the analysis: EPA plus DHA was, in fact, not associated with reduced CV death or non-fatal CV events [243].
In a large meta-analysis, including 50 studies and 294,478 participants, the supplementation of diverse antioxidants, including CoQ10, calcium, zinc, and n-3 fatty acids, did not reduce major CV events vs. no treatment or placebo in both primary and secondary CV prevention. Even in subgroup analyses of the type of intervention, outcome, quality of antioxidant, duration of treatment, and combined vs. single Vitamin administration, no CV benefit was detected, except a slight CV reduction for low-dose Vitamin B6 (RR 0.92, 95% CI from 0.85 to 0.99) [239] (Table 5).
Despite the largely negative RCT data, the Mediterranean diet and fish consumption, known for their antioxidant properties [246], have been associated with a lower risk of CV events or death in large epidemiological studies. Healthy Ageing, a longitudinal study in Europe, including 2239 healthy elderly subjects from two large surveys, followed for a mean of 10 years, showed that the Mediterranean diet was associated with significantly lower risk of all-cause mortality and CV diseases [235] (Table 5). In the Prevención con Dieta Mediterránea (PREDIMED) Study, 7447 subjects at high CV risk but without CV event were assigned to a Mediterranean diet with extra-virgin olive oil integration, a Mediterranean diet with mixed-nuts integration or a dietary fat reduction as control ( Table 5). The primary endpoint of major CV events (MI, stroke, or CV death) was reduced (HR 0.69, 95% CI 0.53-0.92) for the Mediterranean diet with extra-virgin olive oil and for a Mediterranean diet with nuts (HR 0.72; 95% CI: 0.53-0.94) versus the control diet [241]. In the Lyon Diet Heart Study, a secondary prevention trial including 605 subjects with a recent MI, after a mean of 27 months, found that a Mediterranean diet was associated with significantly lower CV death and acute MI [233] (Table 5).
In a meta-analysis, including observational data, comparing regular fish consumption vs. little or no fish intake, fish consumption was associated with a relative risk of 0.83 (95% CI 0.76-0.90) for fatal CAD, and of 0.86 (95% CI 0.81-0.92) for total CHD [247] (Table 5).
Recently, a meta-analysis including data from a large-scale cohort study and three RCTs showed that fish intake (at least 175 g/week) was associated with lower major CV disease, CV, non-CV and total mortality as compared with ≤50 g/month intake [248] (Table 5).

Conclusions
Animal studies strongly support a causal link between some enzymes and systems of generation and/or the clearance of ROS with atherosclerosis development, supporting the notion that controlling ROS is an appropriate goal for therapeutic interventions to prevent ASCVD. However, studies on antioxidant substances in humans led to inconsistent evidence regarding the effect on reducing and preventing ASCVD development or complications to date, while some studies using functional tests or soluble biomarkers have shown a positive impact on the same compounds.
Negative RCTs have helped to identify the pitfalls of the current approaches and how to design future interventions. Problems associated with RCTs can be agent concentrations, exposure time, and ASCVD status (early vs. late), while ROS are not always damaging to cell function since they can also regulate cell homeostasis, and their role is very much celland tissue-dependent. In addition, different ROS may have different roles (H 2 O 2 vs. O 2 •− ). GKT137831 (setanaxib), a promising NOX1/4 inhibitor, is currently in phase II clinical trials for DM kidney disease [249].
In conclusion, while animal models have identified several targets along the paths of ROS production and clearance, intervention RCTs are still lacking, while the dietary habits associated with a possible reduction in ROS tone have shown CV benefits. Future research will have to unravel these gaps, and find the reasons for, and the way to overcome, these inconsistent results.
Funding: Supported in part by institutional funding Linea D1 2020 and Linea d1 2021 to GP and BR.